Magnetic immunosensor and method of use

ABSTRACT

The present invention provides apparatus and methods for the rapid determination of analytes in liquid samples by immunoassays incorporating magnetic capture of beads on a sensor capable of being used in the point-of-care diagnostic field.

CROSS REFERENCE TO RELATED APPLICATION

The present invention claims priority to U.S. Provisional ApplicationNo. 61/371,109, filed on Aug. 5, 2010, the entire contents anddisclosure of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention generally relates to an apparatus and method forrapid determination of analytes in liquid samples by immunoassaysincorporating magnetic capture of beads on a sensor, capable of beingused in the point-of-care diagnostic field, including, for example, useat accident sites, emergency rooms, in surgery, in intensive care units,and also in non-medical environments.

BACKGROUND OF THE INVENTION

A multitude of laboratory immunoassay tests for analytes of interest areperformed on biological samples for diagnosis, screening, diseasestaging, forensic analysis, pregnancy testing and drug testing, amongothers. While a few qualitative tests, such as pregnancy tests, havebeen reduced to simple kits for a patient's home use, the majority ofquantitative tests still require the expertise of trained technicians ina laboratory setting using sophisticated instruments. Laboratory testingincreases the cost of analysis and delays the patient's receipt of theresults. In many circumstances, this delay can be detrimental to thepatient's condition or prognosis, such as for example the analysis ofmarkers indicating myocardial infarction and heart failure. In these andsimilar critical situations, it is advantageous to perform such analysesat the point-of-care, accurately, inexpensively and with minimal delay.

Many types of immunoassay devices and processes have been described. Forexample, a disposable sensing device for measuring analytes by means ofimmunoassay in blood is disclosed by Davis et al. in U.S. Pat. No.7,419,821. This device employs a reading apparatus and a cartridge thatfits into the reading apparatus for the purpose of measuring analyteconcentrations. A potential problem with such disposable devices isvariability of fluid test parameters from cartridge to cartridge due tomanufacturing tolerances or machine wear. U.S. Pat. No. 5,821,399 toZelin discloses methods to overcome this problem using automatic flowcompensation controlled by a reading apparatus having conductimetricsensors located within a cartridge. Each of these patents is herebyincorporated by reference in their respective entireties.

Electrochemical detection, in which the binding of an analyte directlyor indirectly causes a change in the activity of an electroactivespecies adjacent to an electrode, has also been applied to immunoassays.For an early review of electrochemical immunoassays, see Laurell et al.,Methods in Enzymology, vol. 73, “Electroimmunoassay”, Academic Press,New York, 339, 340, 346-348 (1981).

In an electrochemical immunosensor, the binding of an analyte to itscognate antibody produces a change in the activity of an electroactivespecies at an electrode that is poised at a suitable electrochemicalpotential to cause oxidation or reduction of the electroactive species.There are many arrangements for meeting these conditions. For example,electroactive species may be attached directly to an analyte, or theantibody may be covalently attached to an enzyme that either produces anelectroactive species from an electroinactive substrate or destroys anelectroactive substrate. See, M. J. Green (1987) Philos. Trans. R. Soc.Lond. B. Biol. Sci. 316:135-142, for a review of electrochemicalimmunosensors. Magnetic components have been integrated withelectrochemical immunoassays. See, for example, U.S. Pat. Nos.4,945,045; 4,978,610; and 5,149,630, each to Forrest et al. Furthermore,jointly-owned U.S. Pat. No. 7,419,821 to Davis et al. (referenced above)and U.S. Pat. Nos. 7,682,833 and 7,723,099 to Miller et al. teachimmunosensing with magnetic particles.

Microfabrication techniques (e.g., photolithography and plasmadeposition) are attractive for construction of multilayered sensorstructures in confined spaces. Methods for microfabrication ofelectrochemical immunosensors, for example on silicon substrates, aredisclosed in U.S. Pat. No. 5,200,051 to Cozette et al., which is herebyincorporated in its entirety by reference. These include dispensingmethods, methods for attaching biological reagent, e.g., antibodies, tosurfaces including photoformed layers and microparticle latexes, andmethods for performing electrochemical assays.

U.S. Pat. No. 7,223,438 to Mirkin et al. describes a method of formingmagnetic nanostructures by depositing a precursor onto a substrate usinga nanoscopic tip, and then converting the precursor to form a magneticnanostructure. U.S. Pat. No. 7,106,051 to Prins et al. describes amagnetoresistive sensing device for determining the density of magneticparticles in a fluid.

U.S. Pat. Appl. Pub. 2009/0191401 to Deetz et al. is directed tomagnetic receptive paints and coatings that allow magnets to stick tocoated surfaces. These paint and coating compositions containmultiple-sized ferromagnetic particles and a base resin with minimal orno fillers and provide an ultra smooth finish on a substrate. U.S. Pat.No. 5,587,102 to Stern et al. discloses a latex paint compositioncomprising iron particles and U.S. Pat. No. 5,843,329 to Deetz providestechniques for blending magnetic receptive particles into solution formaking magnetic coatings. Jointly-owned U.S. Pat. Nos. 5,998,224 and6,294,342 to Rohr et al. disclose assay methods utilizing the responseof a magnetically responsive reagent to influence a magnetic field toqualitatively or quantitatively measure binding between specific bindingpair members. Each of these patents is hereby incorporated by referencein its entirety.

Both an integrated biosensor for multiplexed immunoassays based onactuated magnetic nanoparticles and a high sensitivity point-of-caretest for cardiac troponin based on an optomagnetic biosensor have beendescribed. See, Bruls et al., Lab Chip 9, 3504-3510 (2009) and Dittmeret al., Clin. Chim. Acta (2010),doi:10.1016/j.cca.2010.03.001,respectively. There are numerousdisclosures of the use of magnetically susceptible particles, e.g., U.S.Pat. No. 4,230,685 to Senyei et al., U.S. Pat. No. 4,554,088 toWhitehead et al., and U.S. Pat. No. 4,628,037 to Chagnon et al. Animportant factor in the use of these particles in assays is efficientmixing to enhance the reaction rate between the target analyte and theparticle surfaces, as opposed to the use of a macro-binding surface thatmainly relies on diffusion. Magnetic mixing systems are disclosed inU.S. Pat. No. 6,231,760 to Siddiqi and U.S. Pat. No. 6,764,859 toKreuwel et al.

Notwithstanding the above literature, there remains a need in the artfor improved immunosensing devices with greater sensitivity for thedetection of analytes, including, for example, cardiac troponin I forearly detection of myocardial infarction. These and other needs are metby the present invention as will become clear to one of skill in the artto which the invention pertains upon reading the following disclosure.

SUMMARY OF THE INVENTION

The present invention is directed to a magnetic immunosensing device andmethods of performing an immunoassay with magnetic immunosensors toprovide diverse real-time or near real-time analysis of analytes.

In one embodiment, the invention is directed to a magnetic immunosensingdevice, comprising: a sensing electrode on a substantially planar chip,wherein the electrode is positioned in a conduit for receiving a samplemixed with antibody-labeled magnetically susceptible beads; and anintegrated high-field permanent magnetic layer on the chip, wherein themagnetic layer is positioned relative to the electrode, therebyattracting the beads substantially proximate to the electrode andsubstantially retaining the beads at the electrode surface duringremoval of unbound sample and washing of the electrode.

Another embodiment of the present invention is directed to amicrofabricated magnetic layer on a substantially planar surface,comprising: high-field permanent magnetic particulates, wherein saidparticulates are dispersed in a thermally, chemically or photoformablycured immobilization matrix; and a microfabricated sensing electrode.

In another embodiment, a method of performing a sandwich immunoassay foran analyte in a sample with a magnetic immunosensor, wherein saidimmunosensor comprises a sensing electrode on a substantially planarchip and an integrated layer on said chip that is magnetized andpositioned substantially proximate to the electrode is provided. Thismethod comprises (a) mixing magnetically susceptible beads coated with acapture antibody to an analyte with a sample containing the analyte anda signal antibody to form a sandwich on said beads; (b) applying themixture to the immunosensor; (c) magnetically localizing and retainingat least a portion of said beads on the electrode; (d) washing theunbound sample from the electrode; (e) exposing the signal antibody ofthe sandwich to a signal generating reagent; and (f) measuring a signalfrom the reagent at the electrode.

In a further embodiment, the invention is directed to a method ofperforming a competitive immunoassay for an analyte in a sample with amagnetic immunosensor, wherein said immunosensor comprises a sensingelectrode on a substantially planar chip and an integrated layer on saidchip that is magnetized and positioned substantially proximate to saidelectrode. This method comprises (a) mixing magnetically susceptiblebeads coated with a capture antibody with a sample containing a firstanalyte and a second analyte, wherein the second analyte is labeled, topermit binding on said beads; (b) applying the mixture to theimmunosensor; (c) magnetically localizing and retaining at least aportion of the beads on the electrode; (d) washing the unbound samplefrom the electrode; (e) exposing the second analyte to a signalgenerating reagent; and (f) measuring a signal from said reagent at theelectrode.

The above summary of the present invention is not intended to describeeach illustrated embodiment or every implementation of the presentinvention. The figures and the detailed description that follow moreparticularly exemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objectives, features and advantages of the presentinvention are described in the following detailed description of thespecific embodiments and are illustrated in the following Figures, inwhich:

FIG. 1 is a cross-sectional illustration of a grooved immunosensor chipin a conduit with applied magnetic fields in accordance with oneembodiment of the present invention;

FIG. 2 is a top view of the grooved immunosensor chip and amicroelectrode array;

FIGS. 3A-C illustrate various exemplary configurations for thepositioning of a rare earth permanent magnet below an immunosensor chipwithin a cartridge;

FIG. 4 is a schematic of magnetic field lines for sensor configurations;

FIG. 5 is a micrograph of magnetically susceptible beads captured on achip surface where the center of the magnet is positioned directly belowthe perimeter of an immunosensor;

FIG. 6 illustrates three traces of the electrochemical detection stepfor the magnetically susceptible bead capture assay: plasma with zerocTnI; Cliniqa control level 3; and Cliniqa control level 4.

FIG. 7 illustrates the positioning of a rare earth permanent magnetbelow an immunosensor chip within a cartridge housing in accordance withone embodiment of the present invention;

FIG. 8 illustrates a foldable cartridge housing in accordance with oneembodiment of the present invention where the rare earth permanentmagnet is positioned underneath an immunosensor chip;

FIG. 9 is a micrograph of magnetically susceptible beads localized on apatterned magnetic layer of PVA and NdFeB particles;

FIG. 10 is a micrograph of a fractured cross-section of the device ofFIG. 9;

FIGS. 11A-C include micrographs of a patterned PVA film with variousparticle sizes of NdFeB (FIG. 11A); ground 6 μm MQP in polyimide (FIG.11B); ground 6 μm MQP in polyimide (FIG. 11C);

FIG. 12A is a micrograph of a 6 μm MQP NdFeB powder and FIG. 12B is amicrograph of the 6 μm MQP NdFeB powder comminuted using a ball mill;

FIG. 13 is a micrograph of beads captured on NdFeB particle surfaces;

FIGS. 14A and 14B show exemplary base immunosensor electrode arrayspartially covered with a printed NdFeB magnetic layer leaving a portionof the perimeter of the array exposed;

FIGS. 15A and 15B depict the over-printed magnetic layer in accordancewith other embodiments of the present invention;

FIG. 16 is a sheared sensor illustrating the printed magnetic layerprofile of FIGS. 15A and 15B;

FIG. 17 illustrates the etched trench process in accordance with oneembodiment of the present invention;

FIG. 18 is a top view of an exemplary underside trench design etchedinto a silicon wafer;

FIG. 19 is the cross-sectional profile of the underside trench;

FIGS. 20A and 20B depict different views of the etched trench;

FIG. 21 shows the etched trench filled with NbFeB powder in a polyimideresin;

FIGS. 22A and 22B are micrographs of a rectangular trench produced on asilicon substrate via reactive ion etching: FIG. 22A shows across-section of the trench and FIG. 22B shows a different cross-sectionof the trench filled with NbFeB powder in a polyimide resin;

FIG. 23 depicts an exemplary combined sensor design for an expandeddetection range where the magnetic zone is comprised a screen printedline of NdFeB powder in a polyimide matrix;

FIG. 24 depicts an exemplary combined sensor design for an expandeddetection range where the magnetic zone is comprised of a bulk NdFeBmagnet;

FIG. 25 depicts another combined sensor design in accordance with oneembodiment of the present invention where the open circles on the chipare potential print locations for the reagents of the present invention;

FIG. 26 is a schematic of an oscillating bead immunoassay (OBIA) with acentral immunosensor flanked by two adjacent magnetic zones with thesmall bead moving in-between in accordance with one embodiment of thepresent invention;

FIG. 27 is a schematic illustrating the degree of bead capture withtime;

FIG. 28 shows a multiplexed OBIA where several different types ofanalyte-capturing beads are present, but where they are effectivelyseparated onto their individual capture sites;

FIG. 29 illustrates the comparative principle of an amperometricimmunoassay for determination of troponin I (TnI), a marker of cardiacinjury;

FIG. 30 is an isometric top view of an immunosensor cartridge cover ofone embodiment of the invention;

FIG. 31 is an isometric bottom view of an immunosensor cartridge coverof one embodiment of the invention;

FIG. 32 is a top view of the layout of a tape gasket for an immunosensorcartridge of one embodiment of the invention;

FIG. 33 is an isometric top view of an immunosensor cartridge base ofone embodiment of the invention;

FIG. 34 illustrates an exemplary segment forming means;

FIG. 35 is a top view of one embodiment of an immunosensor cartridge;and

FIG. 36 is a schematic view of the fluidics of one embodiment of animmunosensor cartridge.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an apparatus and its method of use fordetermining the presence or concentrations of analytes in a liquidsample with a single-use disposable cartridge. The invention is adaptedfor conducting diverse real-time or near real-time assays of analytes.This invention is particularly relevant to high sensitivity cardiactroponin assays in whole blood samples.

In specific embodiments, the invention relates to the determination ofanalytes in biological samples such as blood using magneticelectrochemical immunosensors or other ligand/ligand receptor-basedbiosensors based on a magnetically susceptible bead localization step.The present invention also hereby incorporates by reference in theirrespective entireties jointly-owned U.S. Pat. No. 7,419,821 to Davis etal. and U.S. Pat. Nos. 7,682,833 and 7,723,099 to Miller et al., each ofwhich is referenced above.

One notable advantage of the magnetically susceptible bead captureapproach of the present invention is in improving the low-endsensitivity of immunoassays where there is a perceived benefit indetection of extremely low levels of a marker of myocardial injury(e.g., cTnI). Further advantages and benefits of the embodiments of theinvention described and disclosed herein include but are not limited toease of use, automation of many, if not all steps, of the analysis andelimination of user induced error in the analysis.

I. Magnetic Immunosensor

Various embodiments of the present invention are directed to a magneticimmunosensing device or immunosensor that includes a base sensor orsensing electrode on a substantially planar chip where the sensingelectrode is positioned in a conduit for receiving a sample mixed withbeads that can be attracted to a magnet, or respond to a magnetic field.

A high-field magnet, e.g., a permanent magnet or an electromagnet, ispositioned proximate to the immunosensor chip (e.g., below) orincorporated into the immunosensor chip, for attracting the beads in theconduit substantially proximate to the sensing electrode. This magneticzone functions to substantially retain the beads at or near the sensingelectrode surface during removal of the unbound sample and washing ofthe electrode. As described in detail herein, the beads are coated withan antibody to an analyte in said sample, which provides the basis foran immunoassay. In preferred embodiments, a system comprising a readingapparatus or reader and a single-use cartridge containing the magneticimmunosensor and all the other assay components is used to analyze ananalyte in a biological sample.

A. High-Field Magnet

In certain embodiments of the invention, the magnetic immunosensorcomprises a sensing electrode on a substantially planar chip and has ahigh-field magnet, e.g., a permanent magnet or an electromagnet,positioned proximate to (e.g., below) or associated with the chip. Themagnetic immunosensor of the present invention provides a field ofgreater than about 0.1 Tesla and has an “event horizon” (as definedherein) that can efficiently draw beads from a range of about 0.05 mm toabout 5 mm in the region of the sensing electrode.

The high-field magnet, e.g., permanent magnet or electromagnet, of thepresent invention includes any material that provides a high magneticfield (e.g., greater than about 0.1 Tesla, greater than 0.4 Tesla orgreater than 1 Tesla). The magnetic field can be measured, for example,as the remnant field on a substantially flat surface area of a magnet.While the preferred material is a neodymium iron boron alloy (NdFeB)alloy, and more preferably Nd₂Fe₁₄B, other materials may be used. Forexample, those skilled in the art will recognize that high-fieldpermanent magnets can include ferrite or aluminum nickel cobalt (AlNiCo)magnets, which typically exhibit fields of 0.1 to 1 Tesla. Otherhigh-field permanent magnets comprised of alloys of rare earth elements(e.g., neodymium alloys and samarium cobalt (SmCo) alloys) exhibitfields in excess of 1 Tesla, e.g., greater than 1.2 Tesla or greaterthan 1.4 Tesla.

Rare earth magnets are generally brittle and also vulnerable tocorrosion, and as such these materials are frequently plated or coatedto protect them from breaking and chipping. In addition, the Curie pointof rare earth magnets is substantially above the temperaturesencountered in the immunoassay of the present invention, which may berun in the ambient to about 50° C. range, typically thermostated at 37°C. for assays in blood.

As used herein, “Curie point” or “Curie temperature” refers to acharacteristic property of a ferromagnetic material. The Curie point ofa ferromagnetic material is the temperature above which it loses itscharacteristic ferromagnetic ability to possess a net (spontaneous)magnetization in the absence of an external magnetic field. Attemperatures below the Curie point, the magnetic moments are partiallyaligned within magnetic domains in ferromagnetic materials. As thetemperature is increased from below the Curie point, thermalfluctuations increasingly destroy this alignment, until the netmagnetization becomes zero at and above the Curie point. Above the Curiepoint, the material is purely paramagnetic.

In another embodiment, the high-field magnet comprises an electromagnetin which the magnetic field is produced by the flow of electric current.The electric current may be provided by a reader, in which theimmunosensing device is inserted and with which the immunosensing deviceis in electrical contact.

1. Bulk Magnet Positioned in Housing of Magnetic Immunosensor

The magnetic immunosensor of some embodiments of the invention comprisesa sensing electrode on a substantially planar chip and a bulk permanenthigh-field magnet positioned proximate to the electrode (e.g., below oron the opposite side of the chip). In certain preferred embodiments, thebulk permanent high-field magnet is positioned in the housing (e.g., cutout or trench in the plastic cartridge) of the magnetic immunosensingdevice. Preferably, the bulk permanent high-field magnet is positionedwithin the base of the plastic cartridge housing (e.g., non-coplanarwith the sensing electrode). In other embodiments, the magnet ispositioned adjacent to or within the reading apparatus or reader of theimmunosensing device.

In one embodiment, the bulk high-field permanent magnet is substantiallycylindrical, having a diameter in the range of about 0.1 mm to about 5mm and a length of about 0.1 mm to about 5 mm, and is positioned toyield an “event horizon” (as defined herein) in the conduit suitable forbead capture within a short period of time (e.g., 1-5 minutes). Theconduit generally has a height of about 0.2 mm to about 5 mm and a widthof about 0.2 mm to about 5 mm, and either a uniform or non-uniformcross-sectional area. In other embodiments, the bulk magnet shape may bein the form of a square, rectangle, oval, flake, pyramid, sphere,sub-sphere, or other shaped form.

The method of some embodiments of the invention includes (a) mixingmagnetically susceptible beads coated with a capture antibody with asample suspected of containing an analyte, and a signal antibody to forma sandwich on the beads, (b) applying the mixture to the immunosensorand magnetically localizing and retaining at least a portion of thebeads on the immunosensor, (c) washing the unbound sample from theimmunosensor, and (d) exposing the signal antibody of the sandwich to asignal generating reagent, and measuring a signal from the reagent atthe electrode. In some embodiments, the method can use magneticallysusceptible beads and signal antibodies dissolved from a dry matrix. Inother embodiments, the method operationally relies on step (a) occurringin a first portion of a conduit and step (b) occurring in a secondportion of a conduit where the sensor is located.

2. Magnetized Layer Integral to Magnetic Immunosensing Device

Another embodiment of the present invention includes a magneticimmunosensing device, which comprises a sensing electrode on asubstantially planar chip. The electrode is positioned in a conduit forreceiving a sample mixed with antibody-labeled magnetically susceptiblebeads and a magnetized layer (e.g., microfabricated magnetic layer). Themagnetized layer may be included on (e.g., positioned over, directlyattached, coated or patterned onto any surface of the chip) or embeddedinto the chip (e.g., positioned within the chip, integral to the chip).This configuration attracts the magnetically susceptible beadssubstantially proximate to the electrode and substantially retains themat the electrode during removal of unbound sample and washing of theelectrode.

The magnetized layer preferably is formed from a mobile magneticcomposition, e.g., a slurry, comprising a material capable of sustaininga high-field permanent magnetic field, e.g., a NdFeB alloy, as particlesin an immobilization or support matrix (e.g., a polyimide, polyvinylalcohol (PVA) or thermoplastic equivalent). This slurry is not limitedby viscosity and can include any viscosity suitable for application. Invarious optional embodiments, the mobile magnetic composition has aviscosity ranging from 0.3 to 300,000 CPS, e.g., from 100 to 100,000 CPSor from 1,000 to 10,000 CPS. The magnetic particles in the slurry ofcertain embodiments of the invention have an average particle size from0.01 μm to 100 μm, e.g., from 0.1 μm to 10 μm or from 3 μm to 7 μm.

In addition to polyimide, PVA and thermoplastic polyimide, two-partchemically cured epoxy resins, kapton and the like may be used as thesupport matrix for fixing the magnetic particles to the wafer. Themethods of curing the matrix may be based on a photo-initiated,thermally initiated or chemically initiated process. In certainembodiments, the immobilization matrix is comprised of other photoformedmatrix materials.

As provided above, the slurry can be applied in a variety of locationsin or on the immunosensing device (e.g., to the front side or backsideof a wafer or chip, electrode, housing, reader, etc.). For example, insome embodiments of the invention, the high-field permanent magneticmaterial is applied to the substantially planar chip in a patternedmanner (e.g., using a mask). In certain embodiments, the high-fieldpermanent magnetic material is also applied to a microfabricated sensingelectrode. In other embodiments, the slurry is applied in a layer belowthe sensing electrode.

Prior to the application process, the slurry may or may not bemagnetized. However, after the deposition step, the magnetic layerpreferably is magnetized to provide directionality to the field.

B. Sensing Electrode

The sensing electrode is preferably microfabricated (e.g., anamperometric gold array) on a substantially planar chip (e.g., siliconwafer), as described in the jointly-owned pending and issued patentscited herein (e.g., U.S. Pat. Nos. 5,200,051 and 7,419,821).

C. Magnetically Susceptible Beads

In various embodiments of the invention, the biological sample, e.g.,blood sample, is amended with magnetically susceptible beads. Themagnetically susceptible beads may be comprised of any material known inthe art that is susceptive to movement by a magnet (e.g., permanentmagnet or electromagnet) utilized in or in concert with the device ofthe present invention. As such, the terms “magnetic” and “magneticallysusceptible” with regard to beads can be used interchangeably.

In some embodiments of the invention, the beads include a magnetic core,which preferably is completely or partially coated with a coatingmaterial. The magnetic core may comprise a ferromagnetic, paramagneticor a superparamagnetic material. In preferred embodiments, themagnetically susceptible beads comprise a ferrite core and an outerpolymer coating. However, the magnetic core may comprise one or more ofFe, Co, Mn, Ni, metals comprising one or more of these elements, orderedalloys of these elements, crystals comprised of these elements, magneticoxide structures, such as ferrites, and combinations thereof. In otherembodiments, the magnetic core may be comprised of magnetite (Fe₃O₄),maghemite (γ-Fe₂O₃), or divalent metal-ferrites provided by the formulaMe_(1x)OFe₃+xO₃ where Me is, for example, Cu, Fe, Ni, Co, Mn, Mg, or Znor combinations of these materials, and where x ranges from 0.01 to 99.

Suitable materials for the coating include synthetic and biologicalpolymers, copolymers and polymer blends, and inorganic materials.Polymer materials may include various combinations of polymers ofacrylates, siloxanes, styrenes, acetates, akylene glycols, alkylenes,alkylene oxides, parylenes, lactic acid, and glycolic acid. Biopolymermaterials include starch or similar carbohydrate. Inorganic coatingmaterials may include any combination of a metal, a metal alloy, and aceramic. Examples of ceramic materials may include hydroxyapatite,silicon carbide, carboxylate, sulfonate, phosphate, ferrite,phosphonate, and oxides of Group IV elements of the Periodic Table ofElements.

In other embodiments of the invention, the magnetic beads comprisenon-magnetic substrate beads formed, for example, of a material selectedfrom the group consisting of polystyrene, polyacrylic acid and dextran,upon which a magnetic coating is placed.

In principal, any correctly-sized magnetically susceptible bead capableof being positioned with the high-field magnet of the present inventionmay be utilized, taking into account the dispersability requirements forthe magnetically susceptible beads. In preferred embodiments, at least50 wt. %, e.g., at least 75 wt. %, of the magnetically susceptible beadsare retained at the electrode surface. In some exemplary embodiments,the average particle size of the magnetically susceptible beads mayrange from 0.01 μm to 20 μm, e.g., from 0.1 μm to 10 μm, from 0.1 μm to5 μm or from 0.2 μm to 1.5 μm. As used herein, the term “averageparticle size” refers to the average longest dimension of the particles,e.g., beads, for example the diameter for spherical particles, asdetermined by methods well-known in the art. The particle sizedistribution of the magnetically susceptible beads preferably isunimodal, although polymodal distributions may also be used inaccordance with the present invention. While use of a sphericalmagnetically susceptible bead is preferred, in other embodiments, otherbead shapes and structures, e.g., ovals, sub-spherical, cylindrical andother irregular shaped particles, are within the meaning of the term“beads” and “microparticles” as used herein.

Commercial sources for magnetically susceptible bead preparationsinclude Invitrogen™ (Carlsbad, Calif., U.S.A.) by Life Technologies™,Ademtech (Pessac, France), Chemicell GmbH (Berlin, Germany), BangsLaboratories, Inc.™ (Fishers, Ind.) and Seradyn, Inc. (Indianapolis,Ind.). Many of the commercially available products incorporate surfacefunctionalization that can be employed to immobilize antibodies (e.g.,IgG) on the bead surfaces. Exemplary functionalizations includecarboxyl, amino or streptavidin-modified magnetically susceptible beads.

The magnetically susceptible beads are preferably coated with anantibody to an analyte that is a cardiovascular marker, e.g., cardiactroponin I, troponin T, a troponin complex, human chorionicgonadotropin, BNP, creatine kinase, creatine kinase subunit M, creatinekinase subunit B, creatine kinase MB (CK-MB), proBNP, NT-proBNP,myoglobin, myosin light chain or modified fragments thereof, amongothers. In addition, markers for other indications can be utilized.Further exemplary analytes include, but are not limited to, beta-HCG,TSH, ultra hTSH II, TT3, TT4, FT3, FT4, myeloperoxidase, D-dimer, CRP,NGAL, PSA, LH, FSH, galectin-3, prolactin, progesterone, estradiol,DHEA-S, AFP, CA 125 II, CA 125, CA 15-3, CA 19-9, CA 19-9XR, CEA,thyroxine (T4), triiodothyronine (T3), T-uptake, Tg, anti-Tg, anti-TPO,ferritin, cortisol, insulin, HBsAg, HCV Ag/Ab combo, HCV core Ag,anti-HCV, AUSAB (anti-HBs), CORE, CORE-M, SHBG, iPTH, theophylline,sirolimus, tacrolimus, anti-HAV, anti-HAV IgM, HAVAB, HAVAB-M,HAVAB-M2.0, HAVAB-G, HAVAB 2.0, HAVAB 2.0 Quant, IgM, CMV IgM, CMV IgG,â-2-microglobulin, digitoxin, HBe, anti-HBe, HBeAg, HIV 1/2gO, HIV Ag/Abcombo, testosterone, SCC, vitamin B12, folate, syphilis, anti-HBc,rubella IgG, rubella IgM, homocysteine, MPO, cytomegalovirus (CMV) IgGAvidity, toxo IgG avidity, toxo IgG, toxo IgM, C-peptide, vitamin D,HTLV I/II, total âhCG, progesterone, estradrogen, prolactin, myoglobin,tPSA, fPSA, carbamazepine (CBZ), digoxin, gentamicin, NAPA, phenytoin,phenobarbital, valproic acid, vancomycin, procaine, quinidine,tobramycin, methamphetamine (METH), amphetamine (AMPH), barbituates,benzodiazepine, cannabis, cocaine, methadone, opiates, PCP,acetaminophen, ethanol, salicylates, tricyclics, holoTc, anti-CCP,HbA1c, barbs-U, among others. In certain embodiments of the invention,the antibody is to a low-abundance analyte in the sample. Abbreviatednames above will be familiar to one of ordinary skill in the clinicalanalytical art.

The magnetic immunosensor and methods of the present inventionpreferably also comprise a second antibody, which is a labeled antibody,also referred to herein as a signal antibody. In some embodiments, thelabeled antibody is in the form of a dissolvable dry reagent, which alsomay comprise the magnetically susceptible beads that are employed in thepresent invention, as discussed below. Both the immobilized and labeledantibodies can be monoclonal, polyclonal, fragments thereof andcombinations thereof. In addition, one or more of the antibodies can belabeled with various labels including a radiolabel, enzyme, chromophore,flurophore, chemiluminescent species, ionophore, electroactive speciesand others known in the immunoassay art. Where the second antibody islabeled with an enzyme, it is preferably ALP, horseradish peroxidase, orglucose oxidase. In other embodiments, the analyte is labeled withfluorescein, ferrocene, p-aminophenol, or derivatives thereof.

In certain embodiments, the magnetically susceptible beads are depositedin a suitable region of the magnetic immunosensing device as asuspension in, for example, a mixture of lactitol and DEAE-dextran suchas that supplied by Advanced Enzyme Technologies (Pontypool, GreatBritain). Evaporation of the solvent, usually water, yields a glassydeposit in which the beads are immobilized. The lactitol/DEAE-dextranallows the beads to be regionalized within the device in a mechanicallyand biochemically stable state, but which also rapidly dissolves uponcontact with a sample.

In other embodiments, the magnetically susceptible beads arehomogeneously mixed with the sample. In still other embodiments, themagnetically susceptible beads may be less homogeneously mixed with thesample; however, the intent is to optimize the position andconcentration of the beads relative to the sensing electrode. Thoseskilled in the art will recognize that the magnetically susceptiblebeads of the present invention may be added to the biological sampleprior to introduction into the magnetic immunosensing device, such as,for example, as an integral part of a blood collection device or as astandard manual addition step. However, for the convenience of the userand to assure a quality assay, the magnetically susceptible beads arepreferably included within the device.

In some embodiments of the invention, the sample, e.g., whole bloodsample, is collected and then amended by dissolving a dry reagentcomprising the magnetically susceptible beads into the sample. Anyportion of the immunosensing device may be coated with the dry reagent(e.g., sensing device, conduit, sample entry port, sample holdingchamber). In addition to the magnetically susceptible beads, the dryreagent may further include one or more of: beads for reducing leukocyteinterference, a leukocidal reagent, buffer, salt, surfactant,stabilizing agent, simple carbohydrate, complex carbohydrate and variouscombinations thereof. The dry reagent can also include an enzyme-labeledantibody (e.g., the above-described labeled antibody) to the analyte.

In various embodiments, the magnetically susceptible beads are used toamend the biological sample, e.g., blood, in a first container orlocation, and then the sample is passed to a second container orlocation that includes the capture and signal antibodies. In someembodiments, the magnetically susceptible beads are contained insolution and mixed with the biological sample, and the resulting amendedsample is introduced into the magnetic immunosensing device. Forexample, a blood sample may be mixed with the magnetically susceptiblebeads to form an amended sample, which is then introduced into thedevice. In certain embodiments, the magnetic immunosensing device, e.g.,cartridge, includes a pouch that contains a liquid comprising themagnetically susceptible beads, which may be mixed with a biologicalsample in the device and then processed substantially as describedherein to form an assay (e.g., sandwich assay) for analyte detection.

In other embodiments, electrowetting is employed to mix a first liquidcomprising the magnetically susceptible beads with a liquid biologicalsample, e.g., blood. In one such embodiment, an apparatus may beprovided for manipulating droplets. The apparatus, for example, may havea single-sided electrode design in which all conductive elements arecontained on one surface on which droplets are manipulated. In otherembodiments, an additional surface is provided parallel with the firstsurface for the purpose of containing the droplets to be manipulated.The droplets are manipulated by performing electrowetting-basedtechniques in which electrodes contained on or embedded in the firstsurface are sequentially energized and de-energized in a controlledmanner. The apparatus may allow for a number of droplet manipulationprocesses, including merging and mixing two droplets together, splittinga droplet into two or more droplets, sampling a continuous liquid flowby forming from the flow individually controllable droplets, anditerative binary or digital mixing of droplets to obtain a desiredmixing ratio.

In addition, any immunoassay format known in the art may be modified toinclude the magnetically susceptible beads of the present invention, forexample, by adding the beads in a sample pre-treatment step. Thepretreatment may be accomplished, for example, by incorporating thebeads in a blood collection device, in a separate vessel, or may takeplace in the immunoassay device itself by incorporation of the beads inthe test cycle of the device.

In various embodiments of the invention, the beads are mobile andthereby capable of interacting with an analyte. After binding to theanalyte of interest, magnetic forces are used to concentrate the beadsat the electrode for measurement causing the magnetically susceptiblebeads to be localized to the amperometric electrode for signaldetection. One advantage of using mobile beads according to the presentinvention is that their motion in the sample or fluid acceleratesbinding reactions, making the capture step of the assay faster.

D. Additives

In some embodiments of the invention, additives may be included in themagnetic immunosensing device or used in conjunction with the assay. Incertain embodiments, an anticoagulant can be added. For example, heparinmay be added to improve performance in cases where the sample was notcollected in a heparinized tube or was not properly mixed in aheparinized tube. A sufficient amount of heparin may be added so thatfresh unheparinized blood will remain uncoagulated during the assaycycle of the cartridge, typically in the range of 2 to 20 minutes. Instill other embodiments, one or more of proclin, DEAE-dextran, trisbuffer, and lactitol can be added as reagent stabilizers. In furtherembodiments, a surfactant such as polysorbate 20, also known as Tween®20, can be added to reduce binding of proteins to plastic, which is apreferred material for the cartridge housing of the magneticimmunosensing device. The addition of a surfactant also facilitates theeven coating of reagents on plastic surfaces and minimizes thecrystallization of sugars (e.g., lactitol). In other embodiments of theinvention, an antibacterial agent or biocide (e.g., sodium azide) may beadded to inhibit bacterial growth.

II. Manufacture of Magnetic Immunosensing Device

In one embodiment of the invention, a silicon wafer is thermallyoxidized to form an insulating oxide layer having a thickness of about 1μm. A titanium/tungsten layer is then sputtered onto the oxide layer toa preferable thickness of about 100 Å to about 1000 Å, followed by alayer of gold that is from 500 Å to 1000 Å thick, most preferably about800 Å thick. Next, a photoresist is spun onto the wafer and is dried andbaked. The surface is then exposed using a contact mask, the latentimage is developed, and the wafer is exposed to a gold-etchant. Thepatterned gold layer is coated with a photodefinable polyimide, suitablybaked, exposed using a contact mask, developed, cleaned in an oxygenplasma, and preferably imidized at 350° C. for about 5 hours. Thisleaves a large number of electrode openings in the polyimide layer in asquare array. In some embodiments, the square array has a diameter, forexample, from 2 μm to 100 μm, from 5 μm to 15 μm or about 7 μm, with aninter-distance of, for example, from 5 μm to 100 μm, from 10 μm to 20 μmor about 15 μm. The area covered by these electrodes (i.e., sensor area)is substantially circular with a diameter of, for example, from 50 μm to1000 μm, from 100 μm to 300 μm or about 300 μm.

After dicing the wafer into individual chips, each chip is assembledinto a single-use cartridge. The cartridges may be of the type describedin U.S. Pat. Nos. 7,419,821 to Davis et al. or jointly-owned U.S. PatentApplication No. 61/288,189, entitled “Foldable Cartridge Housings forSample Analysis,” filed Dec. 18, 2009, the entireties of which areincorporated herein by reference. In one embodiment, the sensor ispositioned in a conduit for receiving a sample, and a high-field magnet,e.g., permanent or electromagnet, is positioned directly below thesensor, preferably in the center region thereof. In another embodiment,a high-field magnet can be positioned above the sensor region of theconduit. These elements may be in a fixed position within the instrumenthousing, or adapted to an actuator capable of moving in and out ofposition with respect to the immunosensor and conduit. The one or morehigh-field magnets can be used for attracting magnetically susceptiblebeads in the conduit (e.g., substantially proximate to the sensor) andretaining them in the region of the sensor during removal of sample andwashing of the sensor to remove unbound or partially absorbed reagents.As described above, the magnetic beads are coated with an antibody to ananalyte that may be present in the sample.

A. Trenches

In the immunosensor embodiment of the invention shown in FIG. 1,additional features are depicted. In particular, the cross-sectionalrepresentation of FIG. 1 shows an electrochemical sensor patterned insuitable material (e.g., photoformable polyimide) with trenches (orgrooves) having width of from about 1 μm to about 100 μm, e.g., from 3μm to 50 μm or from 10 μm to 20 μm, height or depth of from about 0.1 μmto about 100 μm, e.g., from 1 μm to 50 μm or from 5 μm to 10 μm, andlength of about 1 μm to about 5000 μm, e.g., from 10 μm to 1000 μm orfrom 100 μm to 500 μm. In this immunosensor design embodiment, theplanar chip is positioned into an analytical system capable of applyingmagnetic fields in either direction in roughly the z axis, as shown byvectors B1 and B2. In some embodiments and as shown in FIG. 1, thetrenches may be oriented in a direction substantially parallel to thedirection of sample flow in the immunosensing device. In alternativeembodiments, the trenches may be oriented in a direction substantiallytransverse to or perpendicular to the direction of sample flow. Thetrench structure of the present invention may beneficially inhibit fluidmotion in the x and/or y directions from removing any magneticallysusceptible beads that have been magnetically localized within a trench,for example, by a washing fluid.

A top view illustration of the electrochemical sensor of FIG. 1 isillustrated in FIG. 2, in which microelectrodes of diameter μD (i.e.,about 1 μm to about 100 μm) and spacing μL (about 5 μm to about 500 μm)are observed at the bottom of each groove in the sensor structure as anarray. These microelectrodes can be comprised of gold or other suitableconductor and can be patterned as described above (e.g., with gold andpolyimide). In preferred embodiments, the high-field magnet ispositioned on the underside or below the electrochemical sensor.

B. Magnetic Fields

In some embodiments of the invention, a sandwich immunoassay is formedon the magnetically susceptible beads substantially everywhere in theconduit and the beads are slowly collected on or adjacent to a sensorsurface by bringing the magnet proximal to the cartridge and slowlyoscillating the fluid. In certain embodiments, the oscillating magneticfields are produced by a moveable high-field magnet, e.g., permanentmagnet or electromagnet, located inside the instrument. In otherembodiments, one or more magnets are stationary inside the instrumentand the sandwich formation is located in a position beyond the field.The sample may then be directed, e.g., by a pump or similar means, intoa region where the magnetic field is sufficiently strong for capture.

In a preferred embodiment, the sample is oscillated in a back and forthmotion over the sensor, e.g., by one or more pumps, while the magneticfield is applied, in order to maximize the opportunity for themagnetically susceptible beads to be attracted to the sensor surface.

III. Methods of Performing Immunoassays

The present invention is applicable to methods of performingimmunoassays with a magnetic immunosensor. In preferred embodiments, thepresent invention may be employed in one or more of the following areas:immunosensors, most notably in the context of point-of-care testing;electrochemical immunoassays; immunosensors in conjunction withimmuno-reference sensors; whole blood immunoassays; single-use cartridgebased immunoassays; and non-sequential immunoassays with only a singlewash step; and dry reagent coatings. As will be appreciated by thoseskilled in the art, the general concept disclosed herein is applicableto many immunoassay methods and platforms.

The methods of the invention are applicable to various biological sampletypes (e.g., blood, plasma, serum, urine, interstitial fluid andcerebrospinal fluid). The present invention is applicable a variety ofimmunoassays including both sandwich and competitive immunoassays.

A. Sandwich Immunoassay

In sandwich assay embodiments, the sample contacts the immunosensor withan immobilized first antibody to the target analyte, and a labeledsecond antibody to said target analyte. In some embodiments of theinvention, the sample, e.g., whole blood sample, is collected and thenamended by dissolving a dry reagent comprising magnetically susceptiblebeads into the sample. As discussed above, the beads preferably includean antibody to the analyte of interest immobilized on the outer surfacethereof. The dissolution of the dry reagents and the sandwich formationstep can occur concurrently or in a stepwise manner.

The magnetically susceptible bead concentration employed may varywidely. In some exemplary embodiments, the sample is amended with themagnetically susceptible beads to provide a dissolved bead concentrationof at least 5 μg per μL of sample, e.g., at least 10 μg per μL ofsample, or at least 15 μg per μL of sample. The dry reagent preferablydissolves into the sample to give a bead concentration of from about 5μg to about 40 μg beads per μL of sample, preferably from about 10 toabout 20 μg beads per μL of sample. Depending on the size of the beads,this corresponds to at least about 10⁴ beads per μL of sample, at leastabout 10⁵ beads per μL of sample, or approximately from about 10⁵ toabout 10⁶ beads per μL of sample. Thus, in some preferred embodiments,the beads are present in an amount sufficient to provide a dissolvedbead concentration of at least 10⁴ beads per μL of sample, e.g., atleast about 10⁵ beads per μL of sample, or from about 10⁵ to about 10⁶beads per μL of sample. Once this step is completed, it is possible toperform an immunoassay, e.g., an electrochemical immunoassay, on theamended sample to determine the concentration of an analyte. Inpreferred embodiments of the invention, at least about 10,000 beads areused for each assay. This lower limit reduces counting error issues,e.g., about 1% or greater for about 1,000 beads or less. Defining anupper limit is less straightforward and depends on bead size, howeverabout 10,000,000 beads is generally sufficient. In certain embodiments,the dissolved bead volume is less than about 1% of the total sampleassay volume, and is preferably less than about 0.1%.

In the actual assay step, in preferred embodiments, once the sandwich isformed between the immobilized and signal antibodies on the outersurface of the magnetically susceptible beads, a magnetic field isapplied to attract the beads in the conduit substantially proximate tothe electrode. The sample is subsequently washed to a waste chamberwhile leaving the retained beads substantially proximate to theelectrode, followed by exposing the sandwich on the magneticallysusceptible beads to a substrate capable of reacting with an enzyme toform a product capable of electrochemical detection. One exemplaryformat is an electrochemical enzyme-linked immunosorbent assay.

In some embodiments of the invention, a magnetized layer (e.g.,microfabricated magnetic layer) can be used as the basis for a sandwichimmunoassay method. For example, in one embodiment, the invention is toa method of performing an immunoassay with a magnetic immunosensor wherethe immunosensor comprises a sensing electrode on a substantially planarchip and an additional layer on the chip that is magnetized, positionedin the region of the electrode. This method comprises (a) mixingmagnetically susceptible beads coated with a capture antibody with asample containing (or suspected of containing) an analyte and a signalantibody to form a sandwich on the beads, (b) applying the mixture tothe immunosensor and magnetically localizing and retaining at least aportion of the beads on the immunosensor, (c) washing the sample andunbound material from the immunosensor and exposing the signal antibodyof the sandwich to a signal generating reagent, and (d) measuring asignal from the reagent at the electrode. Furthermore, step (a)preferably occurs in a first portion of a conduit and step (b)preferably occurs in a second portion of a conduit where the sensorresides. In some embodiments, other features of the assay (e.g., sampletype, the use of dry reagents including magnetically susceptible beadsand the signal antibodies, as described herein) can be optimized tofacilitate the dissolution of a dry matrix into the sample.

B. Competitive Immunoassay

Embodiments of the present invention are also applicable to methods ofperforming a competitive immunoassay with a magnetic immunosensor. Intraditional competitive assay embodiments, a sample contacts animmunosensor comprising an immobilized first antibody to a targetanalyte, and a labeled target analyte that competes for binding with thetarget analyte. Because the (unlabeled) target analyte competes with(amended) labeled target analyte, the resulting signal is inverselyproportional to the native analyte concentration of the sample.

In some such embodiments, the method of the invention includes mixingmagnetically susceptible beads coated with a capture antibody with asample containing (or suspected of containing) an analyte and an addedlabeled form of the analyte to permit competitive binding on themagnetically susceptible beads, applying the mixture to an immunosensorand magnetically localizing and retaining at least a portion of thebeads on the immunosensor, washing the sample and unbound material fromthe immunosensor, exposing the labeled analyte to a signal generatingreagent, and measuring a signal from the reagent at the sensingelectrode.

C. i-STAT® Immunoassay

While the present invention is broadly applicable to immunoassaysystems, it is best understood in the context of the i-STAT® immunoassaysystem (Abbott Point of Care Inc., Princeton, N.J., USA), as describedin the jointly-owned pending and issued patents cited herein.

In these immunoassay systems, only a small fraction of analyte presentin the sample is captured. The assay involves sampling of an analytepresent in plasma or whole blood by capturing analyte on anantibody-labeled microparticle which is coated (or permanently bound) tothe surface of a sensor, e.g. an electrochemical sensor. In certainembodiments, a second antibody labeled with an enzyme then binds to theanalyte to make a sandwich. The electrochemical detection format ensuresthat substantially all the enzyme (e.g., alkaline phosphatase (ALP)) isdetected (i.e., detection efficiency approaches 100%), which allows fora high sensitivity assay. Typically, however, the overall amount ofanalyte that is in the region of the antibody-labeled microparticle andsusceptible to capture during normal usage is low due to mass transportlimitations. It would be advantageous, therefore, to capture a higherpercentage of the analyte, while still retaining the high efficiency ofthe detection step. As disclosed in the present invention, more analyteadvantageously may be captured and detected by employing capture of amagnetically susceptible bead reagent that is distributed (e.g.,homogeneously distributed) throughout the sample during the analytecapture step, but is magnetically localized to the sensor in such a waythat retains the ability to measure the enzyme with sufficientlyimproved detection efficiency.

In certain embodiments, the sample, e.g., plasma or whole blood sample,is amended with interference-reducing and/or conditioning reagentsoptionally located in a sample inlet print of a cartridge. For example,the reagents may contain immunoglobulins, immunoglobulin-coatednon-magnetically susceptible beads, non-magnetically susceptible beadreagents for interference screening, and other stabilizing orconditioning reagents. See, e.g., U.S. patent application Ser. Nos.12/620,230 and 12/620,179, both filed Nov. 17, 2009, each of which isincorporated herein by reference in its entirety, for a description ofthe use of sacrificial beads for reducing or eliminating interferencecaused by leukocytes in a blood sample. See also U.S. patent applicationSer. No. 12/411,325, the entirety of which is incorporated herein byreference, which describes the use of non-human IgM and/or IgG orfragments thereof to reduce interference caused by heterophileantibodies.

Upon being pushed into a conduit, the sample is amended with at leasttwo reagents, a primary capture reagent and a labeled conjugate reagent.The primary capture reagent comprises magnetically susceptible beadscoated with antibodies or antibody fragments appropriate to the analyteof interest. This magnetically susceptible bead reagent may be printed,for example, on a wall portion of the holding chamber or attached to aconduit in a region upstream of the sensor chip, using methods describedin jointly-owned U.S. Pat. No. 5,554,339 to Cozzette et al., which ishereby incorporated by reference in its entirety. The labeled conjugatereagent preferably also comprises a signaling element (e.g., ALP). Thisreagent may be printed, for example, as a dissolvable matrix with theprimary capture reagent in a one stage assay. In another embodiment, thetwo reagents are amended into the sample separately from one another, ineither order.

To promote sandwich formation on the magnetically susceptible beads, itis desirable that the amended sample be oscillated or mixed in theconduit for an appropriate period of time (e.g., about 1 minute to about20 minutes or about 5 to 10 minutes). This allows analyte present in thesample to be captured on the surface of the beads and labeled with asignal-generating conjugate.

In one embodiment, following capture, magnetic field B1 is applied insuch a way that the magnetically susceptible beads are induced tomigrate to the top of the sensor channel, opposite the sensor surface,where they are temporarily retained. The surface (i.e., top of sensorchannel) may be patterned in such a way that there is a tendency of thebeads to resist (beyond the action of B1) movement in the direction ofmixing upon subsequent fluid movement. This process can be accompaniedby low-amplitude oscillations in the direction of the capture motion(x-axis) in order to assist capture of the magnetically susceptiblebeads without entrapping formed elements or non-magnetically susceptiblebeads.

In one embodiment, the sample is then moved to a waste chamber. However,in a preferred embodiment, the sample is moved to a lock-wick feature bymeans of pressurization actuated by an air-bladder as described injointly-owned U.S. Pat. No. 7,419,821 to Davis et al. (referenced above)and U.S. Pat. No. 7,723,099 to Miller et al. (referenced above). Each ofthese patents is hereby incorporated by reference in its entirety.

The movement of the magnetically susceptible beads is conducted in sucha way that the magnetic field B1 is able to temporarily retain the beadsat the ceiling of the sensor channel, preferably substantially oppositethe sensor. During this period, the sensor channel is washed in afashion similar to that described in U.S. Pat. Nos. 7,419,821 and7,723,099 (referenced above). In preferred embodiments, prior tocompletion of the washing step, a portion of the wash fluid is left inthe sensor channel until completion of the following magnetic actuationstep. In this embodiment, the magnetic field B1 is reversed to B2 withthe effect that the beads formerly held in position at the sensorchannel ceiling now migrate towards the trenches or grooved sensorstructure. Low amplitude oscillations of the fluid portion in thedirection of capture mixing can be applied in order to help settle thebeads into the trenches of the immunosensor. Upon completion of thisstep, the wash fluid is slowly pulled from the sensor channel andoptionally the magnetic field B2 is turned off. A current arising fromthe diffusion of an electroactive species generated by the action ofbound label on a suitable electrogenic substrate contained in the washfluid is then measured. (See, for example, U.S. Pat. Nos. 7,419,821 and7,723,099 (referenced above)).

As described above, the trenches or grooved structure of theimmunosensor are intended to aid capture of the magnetically susceptiblebeads and yet allow for efficient washing in the direction of fluidmovement. However, other suitable structures may also be used. Oneexemplary structure includes a grid (e.g., rectilinear) array. In thisembodiment, the ability to retain the beads over the array willgenerally depend on the ability to focus magnetic fields in such a waythat highest field densities are contained within the area demarcated bythe area of the array.

In some embodiments, it is desirable to seek to capture substantiallyall or a reliable fraction (e.g., over 75 wt. %) of the beads, in whichcase the dimensions of the bead retention feature (e.g., trenches) areof secondary importance provided that they supply sufficient volume tocontain the beads. Alternatively, a fixed proportion of the beads may besampled in which case the total volume of the capture feature(s) must beheld constant. Certain embodiments of the invention utilize sequentialapplication of two opposing magnetic fields. In other embodiments, asingle applied field may be utilized. Furthermore, in some embodiments,the ability to provide sufficient substrate to the enzyme-limiteddetection reaction may require high substrate concentrations (e.g.,about 20 mM).

The number and dimensions of the optional trenches or other retentionfeatures are dependent on the size and number of the magneticallysusceptible beads required to achieve efficient capture of analytepresent in the sample. In some embodiments, the length of the trenches,for example, may be several thousand microns while the height and widthmay be on the order of several microns. In addition, one function of thetrenches is to allow for localization and consolidation of the beads andenhanced resistance against fluid motion to dislodge them. In certainembodiments, the beads inside the trenches are mobile.

In other embodiments, after dicing the wafer into individual chips, eachchip preferably is assembled into a single-use cartridge and in thisexample a standard immunosensor is used without the groove features. Thesensor is positioned in a conduit for receiving a sample, wherein asingle high-field magnet, e.g., permanent magnet or electromagnet,preferably a neodymium iron boron magnet (e.g., Nd₂Fe₁₄B), is alsopositioned directly below the center of the sensor. The high-fieldmagnet is used for attracting magnetically susceptible beads in theconduit (e.g., substantially proximate to the sensor) and retaining themin the region of the sensor during removal of sample and washing of thesensor. In preferred embodiments, the beads are coated with an antibodyto an analyte in the sample or suspected of being present in the sample.

FIGS. 3A-C shows three exemplary configurations of the sensor chip ordie in a base or cartridge housing of the type described injointly-owned U.S. Pat. Nos. 7,419,821 and 7,723,099 (referenced above)or U.S. Patent Application No. 61/288,189 (referenced above). The ovalstructure on the sensor die corresponds to the immunosensor, which ispositioned in the conduit. In each embodiment, the high-field permanentmagnets are cylindrical with lengths of from 1 mm to 10 mm, e.g., from 2mm to 5 mm, preferably about 3 mm, and diameters of from 0.1 mm to 5 mm,e.g., from 0.5 mm to 2 mm. In FIGS. 3A-C, the magnets have diameters ofabout 1 mm, about 0.5 mm and about 0.3 mm, respectively. The magnets areabutted to the underside of the chip, which preferably has a thicknessof from about 0.2 mm to 5 mm, e.g., from 0.5 mm to 2 mm or preferablyabout 1 mm. FIG. 4 is a schematic representation of the magnetic fieldlines as the magnet diameter decreases from 1 mm to 0.3 mm andillustrating how the field and magnet selection may impact where on thesensor the magnetically susceptible beads are attracted and focused.

FIG. 5 is a micrograph of magnetically susceptible beads captured on achip surface where the center of the magnet having a diameter 1 mm ispositioned directly below a point on the perimeter of an immunosensor.This configuration assisted with showing the contrast between beads(black), the gold electrode area (white) and the base silicon material(gray). As shown in FIG. 5, a majority of the beads are localized ontothe area of the surface directly above the magnet when a suspension ofthe beads is passed down the conduit and into the region of the magnetfor capture. The beads were about 3 μm in diameter.

An immunoassay for cardiac troponin I (cTnI) was performed with a 0.5 mmdiameter high-field permanent magnet positioned directly under thecenter of an immunosensor. Unlike the embodiments using two magnetsdescribed above, in this embodiment, the bead sandwich formation step isperformed in a portion of the conduit upstream from the sensor, so as toavoid localizing the beads onto the sensor prematurely. FIG. 6 includesthree traces of the electrochemical detection step (chronoamperometry)for the magnetically susceptible bead capture assay: plasma with zerocTnI; Cliniqa control level 3; and Cliniqa control level 4. These bitmapcurves were run in substantially the same way as a commercial cTnIcartridge, but with special software for the new fluidic motions. Allthe reagents for this particular experiment were printed on the sensorchip. The cTnI levels L3 and L4 were about 11 ng/mL and 40 ng/mLrespectively. Each trace shows the current at the electrode as afunction of time. The rise time of the current to a steady-state valuereflects the time constant (TC) for the sensor and the plateau valuereflects the amount of analyte in the sample. The traces in FIG. 6 showonly the detection step, the complete assay took about 12 minutes, whichis acceptable for quantitative point of care immunoassays.

Embodiments of the present invention demonstrate that using a fixedpermanent magnet in the present device can reliably capture beads (e.g.,superparamagnetic or ferromagnetic beads) onto an immunosensor withoutsubstantial agglomeration of the beads. In addition, the cartridgefluidic system disclosed in jointly-owned U.S. Pat. Nos. 7,419,821 and7,723,099 (referenced above) or U.S. Patent Application No. 61/288,189(referenced above) provide a foundation for controlling the sample sothat it is amended with reagents and allowed time to react before itpasses through to the region of the conduit with the magnet. FIG. 7illustrates the positioning of a rare earth permanent magnet R21 belowan immunosensor chip within a cartridge housing in accordance with oneembodiment of the present invention, and FIG. 8 illustrates a foldablecartridge of the type described in previously referenced U.S. PatentApplication No. 61/288,189 (referenced above), where the magnet, e.g.,rare earth permanent magnet (not shown), may be positioned underneathimmunosensor chip 204 or one of the other adjacent chips.

In some embodiments, an immunosensor is provided where the magneticcomponent is directly integrated into sensor manufacture, rather thanbeing a separate component (e.g., bulk permanent high-field magnet)requiring assembly into the test device. For example, in one embodiment,a mixture of photoformable polyvinyl alcohol (PVA) mixed with groundNd₂Fe₁₄B powder was printed onto a wafer using a microdispensingapparatus of the type described in jointly-owned U.S. Pat. No. 5,554,339(referenced above). The printed area was of a diameter of about 400 μm.After exposure to UV light and a wash step, the adhered layer wasexposed to a solution of magnetically susceptible beads of about 3 μmdiameter. The solution was then removed and the surface washed withbuffer. FIG. 9 is a micrograph of a portion of the printed area wherethe relatively smaller ferromagnetic (dark) beads are accuratelylocalized on the patterned magnetic layer. As shown, small areas of therelatively larger and irregular NdFeB (light) particles are observablebelow the beads. FIG. 10 is a micrograph of a fractured cross-section ofthe device in FIG. 9.

FIGS. 11A-C illustrate details of the patterned PVA and polyimide filmswith various particle sizes of NdFeB (FIG. 10A); ground 6 μm Magnaquenchparticles (MQP) in polyimide (FIG. 10B); and ground 6 μm MQP inpolyimide (FIG. 10C), before exposure to the magnetically susceptiblebeads. These types of layers were formed using Magnaquench particles(MQP), which is a NdFeB powder with an average particle size of about 6μm in polyimide or Shipley Photo Resist (SPR).

It was found in accordance with certain embodiments of the inventionthat the about 3 μm to about 7 μm thick magnetic film of FIG. 9 waspartially less effective in terms of capturing the beads than the about30 μm to about 40 μm thick films of FIGS. 11A-C. While the “captureradius” of the embodiment of FIG. 9 was several tens of microns, thecapture radius of the embodiment of FIGS. 11A-C was at least 200 μm. Inpreferred embodiments, the average capture radius of the sensors is lessthan about 500 μm, e.g., less than 300 μm or less than 250 μm. The shapeand position of the magnet as well as its composition aid in thesensitivity and precision of assays using the techniques of the presentinvention.

FIG. 12A is a micrograph of a MQP NdFeB powder with an average particlesize of about 6 μm. FIG. 12B is a micrograph of the MQP NdFeB powder ofFIG. 12A that has been comminuted via ball milling for about 3 days. Insome embodiments, the communited NdFeB powder provides for a morehomogeneous mobile magnetic composition, which may facilitate magneticcapture of the beads. FIG. 13 is a micrograph of beads captured on NdFeBparticle surfaces. Those skilled in the art will recognize that cautionshould be taken during the grinding process to prevent combustion, e.g.,adding 0.1 wt.% sulphur or cooling prior to opening the grindingcontainer.

FIGS. 14A and 14B show exemplary base immunosensor electrode arrays 110partially covered with a printed polyimide and NdFeB particle matrix 111leaving a portion of the perimeter of the array exposed. Also shown isthe base silicon wafer 112 and a conductive line 113 for connecting thearray to the instrument electronics. FIGS. 15A and 15B depict theover-printed magnetic layer in accordance with other embodiments of thepresent invention, where FIG. 15A is a standard microelectrode array andFIG. 15B is a single ring electrode having a width of about 20 μm. FIG.16 is a sheared (i.e., fractured) sensor illustrating the printedmagnetic layer profile of FIGS. 15A and 15B.

It is preferable that the high-field magnet, e.g., permanent orelectromagnet, be within a few tens of microns of the amperometricsensor electrode surface in order to speed up the time constant (TC) ofthe sensor. In addition, it is preferable to size the magnet so that theattraction of the magnetically susceptible beads substantially dominatesany of the potentially disruptive fluidic steps (e.g., washing), thereby“effectively permanently” trapping the reagent beads once captured onthe immunosensor surface. While not being bound by theory, this isequivalent to a concept of an “event horizon” for the permanent magnet.“Effective permanent” capture relates to the attraction of a magneticparticle to the magnet, at the point at which the acceleration of theparticle is greater than any of the potentially “disruptive” fluidmotions (e.g., mixing oscillations and washing). Physical observation ofthis phenomenon with a microscope, therefore, can give a practical roughestimate of the “event horizon” for any given bead, sensor design andfluidic motion. Such information is useful in refining the overall testsystem design to achieve capture of substantially all or a reliablefraction (e.g., over 75 wt. %) of the beads from device to device. Oneintended use of the present invention is in making single-use disposabletest cartridges in large volumes (i.e., greater than a million per year)and each device must perform reproducibly within a given batch, (i.e.,have clinically acceptable precision and accuracy).

By way of example, in a preferred embodiment of a cTnI assay, a 10 μLsegment of blood sample is oscillated over the immunosensor in a conduitof 0.5 mm height using a prototype design adapted from the i-STAT®immunoassay cartridge format. This process is performed with a 4 secondcycle time (i.e., 4 Hertz) and equates to a maximum fluid velocity ofabout 10 cm per second. The magnetically susceptible beads are observedto move at about 10 μm per second in the direction of the magnetic field(microscope, jig and cartridge combination not shown). However, once thebeads are within about 50 μm of the sensor surface, the beads are withinthe “event horizon” and become captured on the surface. The beads are nolonger subject to the influence of the oscillation (i.e., resuspensionof the particle is substantially negligible). Similarly, the trencheddesign of one embodiment of the present invention (with the intentionalfabrication of a stagnant layer) may facilitate the retention of beadsat the surface during this step.

Unlike assays where magnetically susceptible beads are always in contactwith a liquid phase, in certain embodiments of the present invention,one or more intervening air segments are present between the sample andwash fluid in the conduit. (See, for example, U.S. Pat. No. 7,419,821(referenced above)). Surprisingly and unexpectedly, it was found thatthe menisci formed by the air segments provide a greater shear force tothe captured beads as they pass over the sensor than that generated byfluid oscillation where the beads are in constant contact with theliquid phase. It was also found that the slow passage of the menisciover the sensor surface was more disruptive than a relatively fastermotion. The high-field magnets employed in the present invention (e.g.,any material that provides a high magnetic field (e.g., greater thanabout 0.1 Tesla)) are preferable for optimizing movement of themagnetically susceptible beads in relation to the sensor.

Those skilled in the art will recognize that consistently and reliablyretaining the captured beads on the sensor during the wash step isdesirable in the delivery of accurate analytical results. While theaddition of trenches on the sensor may be advantageous, selection of theappropriate field strength was also found to be a significant parameter.Experiments showed that greater than about 75% of the beads wereretained on the surface during the wash step of the methods of thepresent invention. In one exemplary embodiment, the wash fluid comprisesa 0.1 M diethanolamine buffer (pH 9.8), 1 mM MgCl₂, 1.0 M NaCl, 10 mM4-aminophenylphosphate and 10 μM NaI.

With regard to the optimized test system design, the dimensions of thehigh-field magnet are important in that, generally, if the magnet is toosmall, either the time needed to capture the magnetically susceptiblebeads is too long or the stability (i.e., retained capture) during thewashing step will degrade the assay performance. In addition, if themagnet is too deep below the sensor plane, or held too far away from thesensor surface, the force of attraction will be reduced and more diffuseand the magnetic reagents are poorly focused upon capture. Based on thepresent disclosure, those skilled in the art will understand how tooptimize these various requirements for any given system and geometry(e.g., electrode area and position in a conduit).

In some immunosensor embodiments, where the bulk permanent high-fieldmagnet is positioned proximate to the sensing electrode or sensor (e.g.,in the housing of the magnetic immunosensing device), the magnetdiameter or width affects the time constant (TC) of the sensor and howeffectively the sensor detects the signal-generating enzyme labels boundto the magnetic reagents on the magnet surface. Taking this into accountas well as the relatively high topography designs shown in FIGS. 9 and11A-C, vis-à-vis the common desire to perform wafer fabricationprocessing on substantially planar surfaces, the process illustrated inFIG. 17 was developed. FIG. 17 illustrates the etched trench process inaccordance with one embodiment of the present invention, wherein asilicon wafer 1301 with a surface coating of photoresist 1302 is etchedfirst with hydrofluoric acid (HF) and then with hot potassium hydroxide(KOH) or trimethyl ammonium hydroxide (TMAH) to leave a trench ofcontrolled dimensions 1303 (e.g., a depth and width of from about 5 μmto about 200 μm). A slurry of magnetizable particles (e.g., NdFeBpowder) in a thermoplastic matrix (e.g., polyimide) is thenmicrodispensed 1305 or spin-coated 1306 into the trench thereby forminga substantially flat surface co-planar with the wafer 1301. The wafer1301 may be further processed, as described in jointly-owned U.S. Pat.Nos. 7,419,821 and 7,723,099 (referenced above), to provide animmunosensor array over each etched trench on a wafer.

FIG. 18 is a top view of an exemplary underside trench design etchedinto a silicon wafer using a 800 μm×1000 μm mask. FIG. 19 is the afteretch cross-sectional profile of the underside trench, having a depth of50-90% into the silicon wafer. FIGS. 20A and 20B depict different viewsof the etched trench in accordance with one embodiment of the presentinvention and FIG. 21 depicts the trench filled with NbFeB powder in apolyimide resin. FIGS. 22A and 22B are micrographs of a rectangulartrench produced on a silicon substrate via reactive ion etching by INO(Hamilton, Ontario). FIG. 22A shows a cross-section of the trench whileFIG. 22B shows a different cross-section of the trench filled with NbFeBpowder in a polyimide resin. The NbFeB powder was about 6 μm indiameter.

In another embodiment, the silicon wafer is polished on both sides and atrench is etched on one side and filled with magnetic particle materialin a binder matrix. Sensor manufacturing processes in accordance withU.S. Pat. Nos. 7,419,821 and 7,723,099 (referenced above) can then beperformed on the other side of the wafer. This approach has theadvantage of starting the electrode part of the sensor processes on apristine flat surface. The binder matrix deposition step of the magneticzone can optionally be performed as the last step in the overallprocess.

D. Hybrid Immunoassay

In a hybrid test embodiment of the present invention, a currentcommercial cTnI assay (e.g., i-STAT cTnI cartridge) can be modified toinclude a separate but analytically integrated magnetic captureimmunosensor. The prior art sensor covers the detection range of 0.20ng/mL to 36.00 ng/mL, while certain embodiments of the magnetic captureimmunosensor of the present invention cover a lower, but overlappingrange of 0.002 ng/mL to 1.0 ng/mL. One such embodiment is shown in FIG.23, where the magnetic zone is comprised a screen printed line of NdFeBpowder in a polyimide matrix. Another embodiment is shown in FIG. 24,where the magnetic zone is comprised of a bulk NdFeB magnet havingdimensions of 1.5 mm×100 μm×40 μm. Although the magnet appears to bepositioned on the front side of the chip, it is truly positioned on thebackside, and FIG. 24 is meant as a composite to show how the magnet andelectrode are aligned. FIG. 25 depicts yet other exemplary combinedsensor design where the open circles on the chip are potential printlocations for the reagents of the present invention.

In one embodiment of the multiple amperometric magnetic immunosensorformat, the background subtraction disclosed in jointly-owned U.S. Pat.No. 7,723,099 to Miller et al. is performed. U.S. Pat. No. 7,723,099 ishereby incorporated by reference in its entirety. This approach isoptionally applied to both sensors, based on the inclusion of areference immunosensor for the magnetic bar design. In an exemplaryembodiment, the crossover from primary use of the magnetic sensor to thestandard sensor enables a broad analytical range (e.g., from about 50ng/mL to about 0.001 ng/mL for cTnI).

In a preferred embodiment, the use of the overlapping range sensors willinclude an instrument error detection software protocol, which enablesan inconsistency between the cTnI sensor signals to be detected. Forexample, in the presence of sufficient cTnI in a test sample, bothsensors should yield elevated signals, while in the absence of cTnI,both sensors should yield relatively low amperometric signals. Failureof these conditions being detected by the operating software wouldindicate that the analytical result is unreliable. Consequently, theinstrument would suppresses reporting a result and instead indicate tothe user that the test should be re-run with a new cartridge.

In yet another embodiment, the stabilization of the reagents and mode ofprinting enables a quick curing matrix. Print cocktails for the enzymeconjugate include, but are not limited to the enzyme-labeled antibody tothe analyte in a protein stabilizing matrix of less than 30% solids andmore preferably 10% or lower solids. The printed magnetic materials ofthe present invention cure rapidly (i.e., in less than about 7 days).This relatively quick curing time has the advantage of simplifying themanufacturing process, where a short delay between chip manufacture andassembly into a cartridge is desirable.

IV. Oscillating Magnetic Immunoassays

While the disclosure above generally is based on the concept of a staticmagnetic field localized in the region of the immunosensor, analternative methodology is also envisaged where an oscillating magneticfield is used adjacent to the immunosensor.

One oscillating magnetic field embodiment makes critical use of labeledmagnetically susceptible beads containing both a label antibody, and anenzyme or fluorescent marker or any other detectable label, termedherein as a “signaling moiety.” The label may be detected by any meansknown in the art including simple microscopy or a reflectancemeasurement for the beads attached to the capture site using animmunosensing device. In addition, the label can be measured opticallythrough a simple optical density measurement on the capture regions orelectrochemically, e.g., through an enzymatic reaction. Additionaldetection techniques include optical resonators, nuclear magneticresonance (NMR), piezoelectric, pyroelectric, fluorescence,chemiluminescence and surface acoustic wave, among others.

Another embodiment of the invention is to a method of performing asandwich immunoassay for an analyte in a sample with an immunosensor ona substantially planar surface using a means for applying an oscillatingmagnetic field (e.g., an electromagnet and/or a moving fixed magnet withrespect to the surface of the immunosensor). The first step comprisesmixing the magnetically susceptible beads with a sample containing orsuspected of containing an analyte, wherein the beads are coated with anantibody to the analyte and a signal generating moiety. The second steprequires oscillating the beads across the surface of the immunosensorcoated with a second antibody to the analyte, using one of the magneticmeans mentioned above. An antibody sandwich is formed therebyimmobilizing the beads on the immunosensor. The sample is then washedfrom the immunosensor, and the signaling moiety on the immunosensor isdetected.

Various embodiments of the invention are directed to use of the movementof the magnetically susceptible beads along a surface to accelerate thesignal generation of an immunoassay. In one embodiment, the beads areinitially located in a region between two spaced magnetic zones and canmove freely between the two. Oscillation of the magnetic field may startslowly and increase in frequency and the magnetically susceptible beadsare forced to move back and forth across a surface as the field changes.Through this motion, the beads can be captured in the intervening spaceon a capture area of the immunosensor having capture antibodies for aparticular target analyte. In a preferred embodiment, the beads arelabeled with analyte specific antibody or antibodies.

FIG. 26 is a schematic of an oscillating bead immunoassay (OBIA) with acentral immunosensor flanked by two adjacent magnetic zones with themagnetically susceptible beads moving there between, and FIG. 27 showsthe enhanced degree of bead capture with time in accordance with oneembodiment of the present invention. These immunoassay embodiments areamenable to multiplexing, i.e., testing for more than one type ofanalyte, because the capture event is localized by the captureimmunosensor's specificity to a particular analyte. FIG. 28 illustratesa multiplexed OBIA where several different types of analyte-capturingbeads are present, but where they are effectively separated onto theirindividual capture sites (e.g., separate immunosensors).

As shown in FIG. 28, the format of the OBIA can be multiplexed with thespecific capture regions for the different analytes to be measured beingarranged sequentially between the two magnetic concentration areas. Areference sensor (not shown) is also readily applied in this format byhaving a non-specific antibody layer with the series of specific analytecapture sites. In this embodiment, the magnetically susceptible beadsare prepared containing label antibodies thereon and are mixed togetherin controlled ratios, depending on the analytical considerations of theparticular assays within the multiplexed immunoassay cartridge. All ofthe labeled magnetically susceptible beads will then move between thetwo magnet contact zones across each of the specific capture regions.The different analyte-specific magnetically susceptible beads may havethe same label type or, more preferably, will have different types oflabels. Optionally, the different analyte-specific magneticallysusceptible beads can be mixed into the same sample and allowed to reactprior to their attraction to the sensor surface via the positioning ofthe magnets.

Those skilled in the immunosensing art will recognize that this assayformat relies on the capture of the beads as they traverse over thesurface in the presence of the magnet fields generated on either side ofthe capture areas. As such, the sensor signal-to-noise ratio isdependent on making non-specific binding minimal on the surface exceptwhere the specific capture antibodies are deposited. An importantfeature of one embodiment of the invention is, therefore, to employpairs of immunosensors (e.g., electrodes) with indifferent antibodieswhere the second one acts as a reference sensor. Here, the second sensorsignal is subtracted from the first one of the pair. This generalconcept is disclosed in jointly-owned U.S. Pat. No. 7,723,099(referenced above).

In another variant of the present embodiment, the oscillation of thebeads is kept to one side of the capture region for a period of time inorder to allow analyte capture on the immunosensor surface. A furtherrefinement includes a reference sensor to determine the portion of thenon-specific signal generated during the analyte capture part of theassay. The magnetic field is preferably actuated via a coil around twoends of a tube or conduit, similar to, for example, the formation of NMRshim fields. This allows control of the force the magneticallysusceptible beads experience in contacting the surface of the sensor andminimizes nonspecific binding. Here, the coils have several axes toallow x/y and z motion in the conduit. Fixed magnets on a rotating oroscillating platform may be used in alternative embodiments.

EXAMPLES

The present invention will be better understood with reference to thespecific embodiments set forth in the following non-limiting examples.

Example 1 Immunoassay for Determination of Troponin I (TnI)

FIG. 29 illustrates a comparative amperometric immunoassay for thedetermination of troponin I (TnI) 70, a marker of cardiac injury. In oneembodiment, a blood sample, for example, is introduced into the sampleholding chamber of the immunosensing device of the present invention,and is amended by a conjugate molecule 71 comprising alkalinephosphatase enzyme (AP) covalently attached to a polyclonalanti-troponin I antibody (cTnI). This conjugate specifically binds tothe TnI 70 in the blood sample, producing a complex made up of TnI boundto the AP-aTnI conjugate 72. The blood sample is further amended withpolymer beads with a ferrite core 74 coated with a TnI antibody. Themixture is oscillated in a conduit connected to the holding chamber thatgenerates sandwich formation on the bead.

Positioned in the conduit is the sensor chip (or chips), which includesa conductivity sensor used to monitor where the sample is with respectto the sensor chip. The position of the sample segment within theconduit can be actively controlled using the edge of the fluid as amarker. As the sample/air interface crosses the conductivity sensor, aprecise signal is generated that can be used as a fluid position markerfrom which controlled fluid excursions can be executed. The fluidsegment is preferentially oscillated edge-to-edge over the sensor. Theimmunosensor chip is positioned downstream of the mean oscillationposition in the conduit. A bulk magnet 76 is positioned under the sensorchip and draws the magnetically susceptible beads to the immunosensorsurface.

In the present example, the sensor comprises an amperometric electrodeused to detect the enzymatically produced 4-aminophenol from thereaction of 4-aminophenylphosphate with the enzyme label alkalinephosphatase. The electrode is preferably produced from a gold surfacecoated with a photodefined layer of polyimide. Regularly spaced openingsin the insulating polyimide layer define a grid of small gold electrodesat which the 4-aminophenol is oxidized in a 2 electron per moleculereaction.

Substrates, such as p-aminophenol species, can be selected such that thehalf-wave potential (E_(1/2)) of the substrate and product differsubstantially. Preferably, the E_(1/2) of the substrate is substantiallyhigher (i.e., more positive) than that of the product. When thiscondition is met, the product can be selectively electrochemicallymeasured in the presence of the substrate.

The detection of alkaline phosphatase activity in this example relies ona measurement of the 4-aminophenol oxidation current. This is achievedat a potential of about +60 mV versus the Ag/AgCl reference electrode onthe chip. The specific form of detection used depends on the sensorconfiguration. The concentration of the 4-aminophenylphosphate isselected to be in excess, e.g., 10 times the Km value. The analysissolution is 0.1 M in diethanolamine and 1.0 M NaCl, buffered to a pH of9.8. Additionally, the analysis solution contains 0.5 mM MgCl, which isa cofactor for the enzyme. A carbonate buffer may alternatively beutilized.

In various embodiments, the antibodies are selected to bind one or moreof protein, e.g., human chorionic gonadotrophin, troponin I, troponin T,troponin C, a troponin complex, creatine kinase, creatine kinase subunitM, creatine kinase subunit B, myoglobin, myosin light chain, or modifiedfragments thereof. Such modified fragments are generated by oxidation,reduction, deletion, addition or modification of at least one aminoacid, including chemical modification with a natural moiety or with asynthetic moiety. Preferably, these biomolecules bind to the analytespecifically and have an affinity constant for binding of about 10⁷ to10¹⁵ M⁻¹.

The immunosensor was prepared as follows. A silicon wafer was thermallyoxidized to form an insulating oxide layer with a thickness of about 1μm. A titanium/tungsten layer was sputtered onto the oxide layer to apreferable thickness of about 100 Å to about 1000 Å, followed by a layerof gold that is most preferably about 800 Å thick. A photoresist wasthen spin coated onto the wafer and was dried and baked. The surface wasthen exposed using a contact mask, and the latent image was developed.Next, the wafer was exposed to a gold-etchant. The patterned gold layerwas coated with a photodefinable polyimide, suitably baked, exposedusing a contact mask, developed, cleaned in an O₂ plasma, and preferablyimidized at 350° C. for 5 hours. An optional metallization of the backside of the wafer may be performed to act as a resistive heatingelement, such as for example in embodiments where the immunosensor is tobe used in a thermostatted format.

Example 2 Magnetic Immunosensing Device and Method of Use

The present example describes a method of using a magnetic immunosensingdevice in accordance with one embodiment of the invention. As shown inFIGS. 30-33, an unmetered fluid sample was introduced into samplechamber 34 of a cartridge, through a sample entry port 4. Capillary stop25 prevents passage of the sample into conduit 11 at this stage, andconduit 34 is filled with the sample. Lid 2 is closed to prevent leakageof the sample from out of the cartridge. The cartridge is then insertedinto a reading apparatus, such as that disclosed in U.S. Pat. No.5,821,399 to Zelin (referenced above), which is hereby incorporated byreference. Insertion of the cartridge into a reading apparatus activatesthe mechanism which punctures a fluid-containing package located at 42when the package is pressed against spike 38. Fluid is thereby expelledinto the second conduit, arriving in sequence at 39, 20, 12 and 11. Theconstriction at 12 prevents further movement of fluid because residualhydrostatic pressure is dissipated by the flow of fluid via secondconduit portion 11 into the waste chamber 44. In a second step,operation of a pump means applies pressure to air-bladder 43, forcingair through conduit 40, through cutaways 17 and 18, and into conduit 34at a predetermined location 27. Capillary stop 25 and location 27delimit a metered portion of the original sample. While the sample iswithin sample chamber 34, it is optionally amended with a compound orcompounds present initially as a dry coating on the inner surface of thechamber (e.g., antibody-coated magnetically susceptible beads andenzyme-labeled antibody conjugate). The metered portion of the sample isthen expelled through the capillary stop by air pressure produced withinair-bladder 43. The sample optionally is oscillated in order to promoteefficient sandwich formation on the magnetically susceptible beads.Preferably, an oscillation frequency of between about 0.2 Hz and about 5Hz is used, most preferably about 0.7 Hz.

In the next step, the sample is moved forwards along the conduit suchthat the magnetically susceptible beads can become trapped onto thesurface of the magnetic electrode. Subsequently, the sample is ejectedfrom the conduit by further pressure applied to air-bladder 43, and thesample passes to waste chamber 44. A wash step next removesnon-specifically bound enzyme-conjugate from the immunosensor area ofthe conduit. Wash fluid in the second conduit is moved by a pump means43, into contact with the sensors.

The air segment (meniscus) or segments are produced within a conduit byany suitable means, including a passive means, an embodiment of which isshown in FIG. 34 and described in detail in U.S. Pat. No. 7,682,833(referenced above), or an active means including a transient lowering ofthe pressure within a conduit using pump means whereby air is drawn intothe conduit through a flap or valve. The air segment is extremelyeffective at clearing the sample-contaminated fluid from conduit 15. Theefficiency of the rinsing of the sensor region is greatly enhanced bythe introduction of one or more air segments. The leading and/ortrailing edges of air segments are passed one or more times over thesensors to rinse and resuspend extraneous material that may have beendeposited from the sample. Extraneous material includes any materialother than specifically bound analyte or analyte/antibody-enzymeconjugate complex. However, in accordance with various embodiments ofthe invention, the washing or rinsing step is not sufficientlyprotracted or vigorous so as to promote substantial resuspension of themagnetically susceptible beads or dissociation of specifically boundanalyte or analyte/antibody-enzyme conjugate complex from the beads. Formeasurement, a further portion of fluid containing the enzyme substrateis placed over the beads on the immunosensors, and the current orpotential, as appropriate to the mode of operation, is recorded as afunction of time.

FIG. 34 illustrates the construction of a specific means for passivelyintroducing an air segment into the sample fluid. Within the base of theimmunosensor is recess 140 comprising a tapered portion 141 and acylindrical portion 142 that are connected. The tapered portion is influid connection with a hole of similar diameter in the tape gasket(FIG. 32) that separates the base (FIG. 33) and cover (FIGS. 30 and 31)of the assembled immunosensor cartridge. The recess contains anabsorbent material that, upon contact with fluid, withdraws a smallquantity of fluid from a conduit thereby passively introducing an airsegment into the conduit. The volume of the recess and the amount andtype of material within it may be adjusted to control the size of theair segment introduced. Specific absorbent materials include, but arenot limited to, glass filter and a laminate comprising a 3 μm Versapor®filter (i.e., acrylic copolymer membrane cast on a nonwoven nylonsupport) bonded by sucrose to a 60% viscose chiffon layer.

Example 3 Magnetic Immunosensing Device and Method of Use

The present example describes one of the methods of use of a cartridge.In this embodiment, the cartridge includes a closeable valve, locatedbetween the immunosensor and the waste chamber. For a cTnI assay, ablood sample is first introduced into the sample chamber of thecartridge. In the following time sequence, time zero (t=0) representsthe time at which the cartridge is inserted into the cartridge readingdevice. Times are given in minutes. Between t=0 and t=1.5, the cartridgereading device makes electrical contact with the sensors throughelectrical contact pads and performs certain diagnostic tests. Insertionof the cartridge perforates the foil pouch introducing fluid into thesecond conduit, as previously described herein. The diagnostic testsdetermine whether fluid or sample is present in the conduits using theconductivity electrodes, determine whether electrical short circuits arepresent in the electrodes, and ensure that the sensor and ground (e.g.,reference/counter) electrodes are thermally equilibrated to, preferably,37° C. prior to the analyte determination.

Between t=1.5 and t=6.75, a metered portion of the sample, preferablybetween about 4 μL and about 200 μL, more preferably between about 4 μLand about 20 μL, and most preferably about 7 μL, is used to contact thesensor. The edges defining the forward and trailing edges of the sampleare reciprocally moved over the conductivity sensor region at afrequency that is preferably between 0.2 to 5.0 Hz, and is mostpreferably 0.7 Hz. During this time, the enzyme-antibody conjugate andmagnetically susceptible beads dissolve within the sample. The amount ofenzyme-antibody conjugate that is coated onto the conduit is selected toyield a concentration when dissolved that is preferably higher than thehighest anticipated cTnI concentration, and is most preferably six timeshigher than the highest anticipated cTnI concentration in the sample.

Between t=6.75 and t=10.0, the sample is moved to the immunosensor forcapture of the magnetically susceptible beads. As shown in FIGS. 30-33,the sample is moved into the waste chamber via closeable valve 41,wetting the closeable valve and causing it to close. The seal created bythe closing of the valve 41 permits the first pump means to be used tocontrol motion of fluid from conduit 11 to conduit 15. After the valve41 closes and the remaining sample is locked in the post analysisconduit, the analyzer plunger retracts from the flexible diaphragm ofthe pump mean, creating a partial vacuum in the sensor conduit. Thisforces the analysis fluid through the small hole in the tape gasket 31and into a short transecting conduit in the base, 13 and 14. Theanalysis fluid is then pulled further and the front edge of the analysisfluid is oscillated across the surface of the immunosensor chip in orderto shear the sample near the walls of the conduit. The conductivitysensor on the chip is used to control this process.

The efficiency of the wash is optimally further enhanced by introductioninto the fluid of one or more meniscus or air segment. As previouslydescribed, the air segment may be introduced by either active or passivemeans. Fluid is then forcibly moved towards sensor chip by the partialvacuum generated by reducing the mechanical pressure exerted upon paddle6, causing the “T” region of the sensor channel in the vicinity of thetransecting conduit to fill with analysis fluid. The T region of thesensor channel optionally has a higher channel height resulting in ameniscus with a smaller radius of curvature. Further away from the Tregion towards the post-analytical conduit, the conduit height isoptionally smaller. The analysis fluid passively flows from the T regiontowards this low conduit height region, thereby washing the conduitwalls. This passive leak allows further effective washing of the Tregion using a minimal volume of fluid and without displacing themagnetically susceptible beads. In this embodiment, the fluid locatedwithin the second conduit also contains a substrate for the enzyme. Inother embodiments, amendment of the fluid using dried substrate withinthe second conduit may be utilized.

Following the positioning of a final segment of fluid over the sensor,measurement of the sensor response is recorded and the concentration ofanalyte is determined. Specifically, at least one sensor reading of asample is made by rapidly placing over the sensor a fresh portion offluid containing a substrate for the enzyme. Rapid displacement bothrinses away product previously formed, and provides a new substrate tothe electrode. Repetitive signals are averaged to produce a measurementof higher precision, and also to obtain a better statistical average ofthe baseline, represented by the current immediately followingreplacement of the solution over the immunosensor.

Example 4 Magnetic Immunosensing Device

Referring now to FIG. 35, there is shown a top view of a magneticimmunosensor cartridge in accordance with one embodiment of the presentinvention. Cartridge 150 comprises a base and a top portion, preferablyconstructed of plastic. The two portions are connected by a thin,adhesive gasket or thin pliable film. As in previous embodiments, theassembled cartridge comprises a sample chamber 151 into which a samplecontaining an analyte of interest is introduced via sample inlet 152. Ametered portion of the sample is delivered to the sensor chip 153(comprising an integrated magnetic layer) via the sample conduit 154(first conduit) by the combined action of a capillary stop 152,preferably formed by a 0.012″ laser cut hole in the gasket or film thatconnects the two portions of the cartridge, and an entry point 155located at a predetermined point within the sample chamber whereby airis introduced by the action of a pump means, such as a paddle pushingupon a sample diaphragm 156. After magnetic capture of the beads on theimmunosensor, the sample is moved to vent 157, which contains a wickingmaterial that absorbs the sample and thereby seals the vent closed tothe further passage of liquid or air. The wicking material is preferablya cotton fiber material, a cellulose material, or other hydrophilicmaterial having pores. It is important in the present application thatthe material is sufficiently absorbent (i.e., possesses sufficientwicking speed) that the valve closes within a time period that iscommensurate with the subsequent withdrawal of the sample diaphragmactuating means, so that the sample is not subsequently drawn back intothe region of the immunosensor.

In specific embodiments of the invention, there is provided a washconduit (second conduit) 158, connected at one end to a vent 159 and atthe other end to the sample conduit at a point 160 of the sample conduitthat is located between vent 157 and immunosensor chip 153. Uponinsertion of the cartridge into a reading apparatus, a fluid isintroduced into conduit 158. Preferably, the fluid is present initiallywithin a foil pouch 161 that is punctured by a pin when an actuatingmeans applies pressure upon the pouch. There is also provided a shortconduit 162 that connects the fluid to conduit 154 via a small openingin the gasket 163. A second capillary stop initially prevents the fluidfrom reaching capillary stop 160, so that the fluid is retained withinconduit 158.

After vent 157 has closed, the pump means is actuated, creating alowered pressure within conduit 154. Air vent 164, preferably comprisinga small flap cut in the gasket or a membrane that vibrates to provide anintermittent air stream, provides a means for air to enter conduit 158via a second vent 165. The second vent 165 preferably also containswicking material capable of closing the vent if wetted, which permitssubsequent depression of sample diaphragm 156 to close vent 165, ifrequired. Simultaneously with the actuation of sample diaphragm 156,fluid is drawn from conduit 158, through capillary stop 160, intoconduit 154. Because the flow of fluid is interrupted by air enteringvent 164, at least one air segment (e.g., a segment or stream ofsegments) is introduced.

Further withdrawal of sample diaphragm 156 draws the liquid containingat least one air segment back across the sensing surface of sensor chip153. The presence of air-liquid boundaries within the liquid enhancesthe rinsing of the sensor chip surface to remove remaining sample.Preferably, the movement of the sample diaphragm 156 is controlled inconjunction with signals received from the conductivity electrodeshoused within the sensor chip adjacent to the analyte sensors. In thisway, the presence of liquid over the sensor is detected, and multiplereadings can be performed by movement of the fluid in discrete steps.

It is advantageous in this embodiment to perform analyte measurementswhen only a thin film of fluid coats the magnetically susceptible beadson the immunosensors, ground chip 165, and a contiguous portion of thewall of conduit 154 between the sensors and ground electrode. A suitablefilm is obtained by withdrawing fluid by operation of the samplediaphragm 156, until the conductimetric sensor located next to thesensor indicates that bulk fluid is no longer present in that region ofconduit 154. It has been found that measurement can be performed at verylow (nA) currents, and the potential drop that results from increasedresistance of a thin film between ground chip and sensor chip (comparedto bulk fluid) is not significant.

The ground chip 165 is preferably a silver/silver chloride referenceelectrode and acts effectively as both a counter and reference electrodein an amperometric measurement. It is advantageous, in this embodimentof the invention, to avoid air segments, which easily form upon therelatively hydrophobic silver chloride surface, to pattern the groundchip as small regions of silver/silver chloride interspersed with morehydrophilic regions, such as a surface of silicon dioxide. Thus, apreferred ground electrode (counter and reference electrode combined)configuration comprises an array of silver/silver chloride squaresdensely arranged and interspersed with silicon dioxide. There is afurther advantage in the avoidance of unintentional segments if theregions of silver/silver chloride are somewhat recessed.

Referring now to FIGS. 7 and 36, there is shown a schematic view of thefluidics of the preferred embodiment of an immunosensor cartridge.Regions R1-R7 represent specific regions of the conduits associated withspecific operational functions. In particular, R1 represents the samplechamber; R2 the sample conduit whereby a metered portion of the sampleis transferred to the capture region, and in which the sample isoptionally amended with a substance coated upon the walls of the conduit(e.g., magnetically susceptible beads with antibody to the analyte andantibody conjugate); R21 represents a region for mixing and sampleoscillation; R3 represents the magnetic capture region, which houses theimmunosensors; R4 and R5 represent portions of the first conduit thatare optionally used for further amendment of fluids with substancescoated onto the conduit wall, whereby more complex assay schemes areachieved; R6 represents the portion of the second conduit into whichfluid is introduced upon insertion of the cartridge into a readingapparatus; R7 comprises a portion of the conduit located betweencapillary stops 160 and 166 (shown in FIG. 35), in which furtheramendment can occur; and R8 represents the portion of conduit 154located between point 160 and vent 157, which can further be used toamend liquids contained within.

Example 5 Magnetic Immunosensor System

This example addresses the coordination of fluidics and analytemeasurements as a system. In the analysis sequence, a user places asample into the cartridge, places the cartridge into the analyzer and inabout 1 minute to about 20 minutes, a quantitative measurement of one ormore analytes is performed. Referring to FIGS. 35 and 36, the followingis a non-limiting example of a sequence of events that occur during theanalysis:

1) A 25 to 50 μL sample is introduced in the sample inlet 167 and fillschamber 151 to capillary stop 152 formed by a 0.012″ laser cut hole inthe adhesive tape holding the cover and base components together. Theuser then seals the inlet and places the cartridge into the analyzer.Magnetically susceptible beads having antibodies to the analyte ofinterest and optionally a labeled conjugate antibody to the analyte maybe provided in a dry coating on the walls of chamber 151 such that theydissolve into the sample once the sample is introduced therein. In analternative embodiment, either or both the magnetically susceptiblebeads and the labeled conjugate antibody may be provided in a drycoating within conduit 154.

2) The analyzer makes contact with the cartridge, and a motor drivenplunger presses onto the foil pouch 161, forcing the wash/analysis fluidout into a central conduit 158.

3) A separate motor driven plunger contacts the sample diaphragm 156,pushing a measured segment of the sample along the sample conduit (fromreagent region R1 to R2 and R21). The sample position is detected viaone or more conductivity sensors. The immunosensor chip is located incapture region R3.

4) The sample is oscillated by means of the sample diaphragm 156 in theR21 region in a predetermined and controlled fashion for a controlledtime to promote binding of analyte to the magnetically susceptible beadsand to the antibody conjugate.

5) The sample is pushed towards the waste region of the cartridge (R8)and comes in contact with a passive pump 157 in the form of a celluloseor similar absorbent wick. The action of wetting this wick seals thewick to air flow, thus eliminating its ability to vent excess pressuregenerated by the sample diaphragm 156. The active vent thereby becomesthe “controlled air vent” of FIG. 36.

6) Rapid evacuation of the sample conduit (effected by withdrawing themotor driven plunger from the sample diaphragm 156) forces a mixture ofair (from the vent) and wash/analysis fluid from the second conduit tomove into the inlet located between R5 and R4. By repeating the rapidevacuation of the sample conduit, a series of air separated fluidsegments are generated, which are pulled across the sensor chip towardsthe sample inlet (from R4 to R3 to R21 to R2 and R1). This processwashes the sensor free of excess reagents and wets the sensor withreagents appropriate for the analysis. In certain embodiments, thewash/analysis fluid that originates in the foil pouch can be furtheramended by addition of reagents in R7 and R6 within the centralwash/analysis fluid conduit.

7) The wash/analysis fluid segment is drawn at a slower speed towardsthe sample inlet to yield an immunosensor chip with the retainedmagnetically susceptible beads, which contains only a thin layer of theanalysis fluid. The electrochemical analysis is performed at this point.The preferred method of analysis is amperometry, but potentiometry orimpedance detection is also used.

8) The mechanism retracts, allowing the cartridge to be removed from theanalyzer.

While the present invention has been described in terms of variouspreferred embodiments, those skilled in the art will recognize thatmodifications, substitutions, omissions and changes can be made to suchembodiments without departing from the spirit and scope of the presentinvention.

What is claimed is:
 1. A microfabricated magnetic layer on asubstantially planar surface, wherein the microfabricated magnetic layercomprises: high-field permanent magnetic particulates dispersed in athermally, chemically or photoformably cured immobilization matrix;wherein the immobilization matrix comprises a polyimide, polyvinylalcohol, or epoxy resin; wherein the microfabricated magnetic layer ispositioned substantially proximate to a microfabricated sensingelectrode, wherein the electrode is connected by a conductive line toinstrument electronics, wherein the microfabricated magnetic layer isnot the conductive line, wherein the microfabricated magnetic layer ispositioned to attract magnetically susceptible beads coated with acapture antibody to a target analyte, wherein the electrode is anamperometric electrode, and wherein the magnetic layer is configured toconcentrate the magnetically susceptible beads with the capture antibodyand the target analyte within a sample fluid at the electrode formeasurement, and wherein the amperometric electrode is configured todetect the target analyte.
 2. The microfabricated magnetic layer ofclaim 1, wherein the magnetic particulates comprise a neodymium ironboron (NdFeB) alloy.
 3. The microfabricated magnetic layer of claim 1,wherein the magnetic particulates comprise a Nd₂Fe₁₄B alloy.
 4. Themicrofabricated magnetic layer of claim 1, wherein the magneticparticulates have an average particle size of from 0.01 μm to 20 μm. 5.The microfabricated magnetic layer of claim 1, wherein the magneticparticulates have an average particle size of from 0.01 μm to 5 μm. 6.The microfabricated magnetic layer of claim 1, wherein theimmobilization matrix comprises polyimide.
 7. The microfabricatedmagnetic layer of claim 1, wherein the immobilization matrix comprisespolyvinyl alcohol.
 8. The microfabricated magnetic layer of claim 1,wherein the magnetic particulates provide a magnetic field of greaterthan about 0.1 Tesla.
 9. The microfabricated magnetic layer of claim 1,wherein the microfabricated magnetic layer is positioned to yield anevent horizon for the magnetically susceptible beads in the range ofless than about 200 μm in the region of the electrode.
 10. Themicrofabricated magnetic layer of claim 1, wherein the electrode is agold microarray.
 11. The microfabricated magnetic layer of claim 1,wherein the microfabricated magnetic layer is positioned over thesubstantially planar surface.
 12. The microfabricated magnetic layer ofclaim 1, wherein the microfabricated magnetic layer is directly attachedto the substantially planar surface.
 13. The microfabricated magneticlayer of claim 1, wherein the microfabricated magnetic layer is coatedonto the surface of the substantially planar surface.
 14. Themicrofabricated magnetic layer of claim 1, wherein the microfabricatedmagnetic layer is patterned onto the surface of the substantially planarsurface.
 15. The microfabricated magnetic layer of claim 1, wherein themicrofabricated magnetic layer is positioned proximate to the sensingelectrode.